Titanium-based implant with antibacterial and osteogenesis promoting performance and preparation method and application thereof

Abstract

The invention discloses a titanium-based implant with antibacterial and osteogenesis-promoting properties as well as a preparation method and application thereof, and belongs to the technical field of crossing of biomedical materials and biomedicine. The selective laser melting porous titanium alloy is used as a matrix, micro-arc oxidation and hydro-thermal treatment are utilized, and the multifunctional composite coating with the antibacterial function and the osteogenesis promoting function is constructed on the surface of the selective laser melting porous titanium alloy; the microstructure and the biological performance of the coating are regulated and controlled by doping functional elements, so that the antibacterial performance and the osteogenesis promoting performance of the coating are optimized.

Description

Technical Field

[0001] The present invention belongs to the cross - technical field of biomedical materials and biomedicine, and particularly relates to a titanium - based implant with both antibacterial and osteogenic properties, its preparation method and application. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Medical titanium alloy (Ti6Al4V) is one of the widely used metal implant materials in the current orthopedic and dental fields due to its good mechanical properties, chemical stability and biocompatibility. The lack of bioactivity and active bactericidal effect of titanium alloy makes the implant only mechanically interlocked with bone tissue, which easily causes implant loosening and related infection problems. With the increasing aging of the global population and the gradual increase in bone defects caused by orthopedic diseases and traumas, the clinical demand for bone implants is increasing. Therefore, studying titanium alloy implants with both antibacterial and osteogenic properties to solve the problems of bacterial infection and insufficient bone integration faced by titanium alloy implants has positive significance for promoting the research and application of titanium - based implants.

[0004] New breakthroughs have been made in 3D - printed titanium alloy implants. In September 2023, the hydroxyapatite (HA) - coated porous titanium alloy intervertebral fusion device prepared by Dimensional Bio using selective laser melting (SLM) and vacuum plasma spraying technology was approved for marketing, becoming the world's first approved 3D - printed porous titanium alloy intervertebral fusion device with a bioactive coating. Selective laser melting (SLM) forms complex three - dimensional structures in a layer - by - layer incremental manner. By adjusting parameters such as pore size, shape and distribution, the compressive strength, elastic modulus, etc. of the material are changed, so that the pore structure and mechanical properties of porous metal are approximately matched with bone tissue. However, porous - structured titanium alloys also lack antibacterial and bioactivity. Therefore, considering the overall modification of porous titanium alloys, it is of great significance to provide a method to construct a functional coating on its surface to endow it with antibacterial and osteogenic properties. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a titanium - based implant with both antibacterial and osteogenic properties, its preparation method and application. First, a porous titanium alloy is used as the substrate, and a micro - arc oxidation coating is prepared by micro - arc oxidation technology, and its biological properties are optimized by adding different functional elements to the electrolyte. Then, the micro - arc oxidation - treated porous titanium alloy is used as the substrate, and a multifunctional composite coating with both antibacterial and osteogenic properties is prepared by hydrothermal treatment, and its biological properties are optimized by adding different functional elements to the hydrothermal reaction medium.

[0006] The technical idea of ​​the present invention is: first use micro-arc oxidation treatment + element doping to prepare a coating on the substrate to see whether an implant with both antibacterial and osteopromoting properties can be prepared. However, studies have found that it is difficult to achieve a balance between antibacterial properties and cell activity by introducing functional element doping during micro-arc oxidation. Therefore, subsequent attempts were made to further introduce trace functional elements through hydrothermal treatment. Finally, the substrate was subjected to micro-arc oxidation and hydrothermal treatment in turn, and trace functional elements were introduced during the hydrothermal treatment, thereby achieving the construction of a multifunctional coating with both antibacterial and osteopromoting properties.

[0007] Micro-arc oxidation relies on the instantaneous high temperature and high pressure generated by arc discharge in the electrolyte to grow a metal oxide-based ceramic film on the surface of titanium alloy. The micro-arc oxidation process is mainly affected by the substrate material, power supply parameters, electrolyte composition, reaction time, etc. By adjusting the preparation process parameters, the purpose of regulating the coating composition, structure and performance can be achieved. Micro-arc oxidation can introduce active ions such as calcium and phosphorus into the coating. Micro-arc oxidation can improve the biological activity of metal materials, but the effect is not particularly ideal. Micro-arc oxidation technology is combined with hydrothermal treatment. Hydrothermal treatment can effectively promote the dissolution and recrystallization of reactants through a high temperature and high pressure environment. After subsequent treatment, elements such as calcium and phosphorus in the coating can be crystallized into hydroxyapatite, thereby improving the biological activity of the coating.

[0008] The present invention uses selective laser melting of porous titanium alloy as a matrix, and utilizes micro-arc oxidation and hydrothermal treatment to construct a multifunctional composite coating on its surface that takes both antibacterial and osteogenic properties into account; and regulates the microstructure and biological properties of the coating by doping functional elements, thereby optimizing the antibacterial and osteogenic properties of the coating.

[0009] In order to achieve the above object, the technical solution of the present invention is:

[0010] In a first aspect, the present invention provides a titanium-based implant having both antibacterial and osteogenic properties, comprising a porous titanium alloy substrate and a coating formed on the porous titanium alloy substrate;

[0011] Wherein, the coating is a composite coating doped with functional elements obtained by micro-arc oxidation and hydrothermal treatment;

[0012] The functional element is one or more of strontium, copper, zinc, magnesium and silver.

[0013] Preferably, the functional elements are strontium and copper.

[0014] The atomic ratio of O:Al:Si:P:Ca:Ti:Sr:Cu in titanium-based implants is

[0015] (55 - 70):(0.5 - 3):(1 - 6):(5 - 15):(4 - 16):(3 - 25):(0.5 - 4):(0.01 - 3), preferably (60 - 70):(0.5 - 2):(1 - 2):(5 - 15):(5 - 16):(3 - 25):(0.5 - 4):(0.1 - 0.5), more preferably (60 - 65):(1 - 2):(1 - 1.5):(5 - 10):(5 - 10):(20 - 25):(0.5 - 1):(0.1 - 0.2).

[0016] The functional element doping is doping the functional element during the micro - arc oxidation process and / or during the hydrothermal treatment process, preferably doping the functional element during the hydrothermal treatment process.

[0017] In a specific embodiment, the element doping is doping the functional element during the micro - arc oxidation process. Specifically, during the micro - arc oxidation treatment of the porous titanium alloy, the functional element is added to the electrolyte.

[0018] In a specific embodiment, the element doping is doping the functional element during the micro - arc oxidation process and doping the functional element during the hydrothermal treatment process. Specifically, during the micro - arc oxidation treatment of the porous titanium alloy, the functional element is added to the electrolyte; and during the further hydrothermal treatment, the functional element is added to the reaction medium.

[0019] In a specific embodiment, the element doping is doping the functional element during the hydrothermal treatment process. Specifically, during the micro - arc oxidation treatment of the porous titanium alloy, the functional element is not added to the electrolyte, but during the further hydrothermal treatment, the functional element is added to the reaction medium.

[0020] The thickness of the micro - arc oxidation coating is 30 - 50μm.

[0021] The pore diameter of the porous titanium alloy is set at 200 - 700μm, the rod diameter is set at 200μm - 400μm. By adjusting the proportion of the solid part, the porosity is controlled at 60 - 90%, and porous titanium alloys with different pore parameters are designed. Use computer - aided design software to design the porous structure model, import the STL - format file into the computer, and generate a scan path file to import into the control software of the printer. Use the SLM processing method in a laser printer to prepare the porous titanium alloy. Specifically as follows: laser power 100 - 200W, scan speed 800 - 1500mm / s, scan spacing 50 - 100μm, layer thickness 20 - 40μm; preferably laser power 160W, scan speed 1100mm / s, scan spacing 80μm, layer thickness 30μm.

[0022] Second aspect, the present invention provides a preparation method of the above-mentioned titanium-based implant with both antibacterial and osteogenic properties, including the following steps:

[0023] Using the pretreated porous titanium alloy as the substrate, a porous titanium alloy loaded with a micro-arc oxidation coating is obtained by micro-arc oxidation treatment;

[0024] Using the porous titanium alloy loaded with the micro-arc oxidation coating as the substrate, a composite coating is prepared by hydrothermal treatment to complete the preparation of the titanium-based implant with both antibacterial and osteogenic properties.

[0025] In one or more embodiments, the pretreatment includes cleaning, polishing, and heat treatment of the porous titanium alloy.

[0026] The cleaning is ultrasonic cleaning; the polishing is chemical polishing treatment; the heat treatment is annealing treatment at 700 - 900 °C for 1 - 4 h. The specific steps are as follows: After the selective laser melting formed sample is ultrasonically cleaned, it is chemically polished to clean the surface contamination layer and residual powder of the porous titanium alloy, and then air-dried for standby. The chemically polished sample is placed in a vacuum box furnace and annealed at 700 - 900 °C for 1 - 4 h, and then stored for standby.

[0027] During the 3D printing process, the titanium alloy undergoes complex thermal cycles, which will generate large residual stresses. Annealing treatment can eliminate stress concentration and make the surface state of the material more uniform. If the annealing treatment is not carried out, the residual stresses during the printing process cannot be released, and in the subsequent processing or use process, it will fail due to stress concentration.

[0028] In one or more embodiments, the micro-arc oxidation treatment includes: Connecting the pretreated porous titanium alloy as the working electrode to the anode of the micro-arc oxidation power supply, connecting the stainless steel sheet to the cathode of the power supply, and placing them in the electrolyte.

[0029] In one or more embodiments, the micro-arc oxidation adopts a constant voltage mode, with a current density of 0.05 - 0.15 A / cm 2 , a positive voltage of 400 - 450 V, a negative voltage of 0, a positive pulse number of 1, a negative pulse number of 1, a positive duty cycle of 25 - 35%, a negative duty cycle of 15 - 25%, a frequency of 500 - 700 Hz, and a time of 10 - 15 min.

[0030] In one or more embodiments, the electrolyte includes a calcium source, a phosphorus source, and an auxiliary additive. The calcium source is 0.10 - 0.20 mol / L calcium silicate (CaSiO 3 ), the phosphorus source is 0.005 - 0.015 mol / L sodium hexametaphosphate (NaPO 3 ), 6 , the auxiliary additive includes 4 - 6 g / L sodium hydroxide (NaOH), 4 - 6 ml / L glycerol (C3 H 8 O 3 )。

[0031] Furthermore, the electrolyte further includes an additive containing a functional element. The additive containing a functional element includes strontium chloride and / or copper acetate. The concentration of strontium chloride (SrCl 2 ·6H 2 O) and copper acetate (C 4 H 6 CuO 4 ) is 1 - 6 mmol / L for both.

[0032] In one or more embodiments, the hydrothermal treatment is as follows: placing the porous titanium alloy loaded with the micro - arc oxidation coating in a reaction medium and reacting at 170 - 190 °C for 4 - 8 h.

[0033] In one or more embodiments, the reaction medium includes a reaction basic medium. The reaction basic medium includes water, and the pH of the aqueous solution is adjusted to 9 - 11 using an alkali. The alkali is ammonia water.

[0034] Furthermore, the reaction medium further includes an additive containing a functional element; the additive containing a functional element includes strontium chloride and / or copper acetate.

[0035] The ratio of water, strontium chloride, and copper acetate is (40 - 60 mL):(0.05 - 0.30 mmol):(0.05 - 0.30 mmol), preferably 50 mL:(0.05 - 0.2 mmol):(0.05 - 0.15 mmol).

[0036] In one or more embodiments, after the hydrothermal treatment, it further includes cooling to room temperature, taking out the sample, and ultrasonically cleaning and drying it with water and alcohol in sequence and then setting it aside for use. The alcohol is absolute ethanol.

[0037] In a third aspect, the present invention provides an application of the above - mentioned titanium - based implant with both antibacterial and osteogenic properties or the titanium - based implant with both antibacterial and osteogenic properties obtained by the above - mentioned preparation method in the field of bone tissue repair and bone defect filling.

[0038] One or some of the above - mentioned technical solutions have the following advantages or beneficial effects:

[0039] (1) In view of the lack of antibacterial and osteogenic properties in titanium alloys, which easily cause loosening of implants and related infection problems, the present invention provides a method for preparing a porous titanium-based implant with both antibacterial and osteogenic properties by combining micro-arc oxidation technology and hydrothermal treatment. First, a porous titanium alloy prepared by selective laser melting technology is used as the substrate, and a multifunctional composite coating that takes into account antibacterial and osteogenic properties is constructed on its surface by micro-arc oxidation technology and hydrothermal treatment, and its biological properties are optimized by doping with functional elements.

[0040] (2) Regarding the doping of functional elements, compared with the micro-arc oxidation coating obtained by doping functional elements during the micro-arc oxidation process (doping functional elements in micro-arc oxidation) and the multifunctional composite coating obtained by doping functional elements during the micro-arc oxidation process and further doping functional elements during the hydrothermal treatment process (doping functional elements in micro-arc oxidation + doping functional elements in hydrothermal treatment), the composite coating obtained by adding functional elements only during the hydrothermal treatment (no doping of functional elements in micro-arc oxidation + doping of functional elements in hydrothermal treatment) can achieve both high antibacterial and osteogenic performance effects.

[0041] (3) The preparation method of the porous titanium-based implant with both antibacterial and osteogenic properties disclosed by the present invention solves the problems such as bacterial infection and insufficient bone integration faced by titanium-based implants, provides high-quality materials for bone tissue repair and bone defect filling, ensures the safety and reliability of implant materials, and has positive significance for promoting the research and application of titanium-based implants. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0043] Figure 1 are the surface morphologies of the metal implants in Example 1, Example 2, and Example 6 of the present invention; wherein, (a) is the surface morphology of the metal implant in Example 1, (b) is the surface morphology of the metal implant in Example 2, and (c) is the surface morphology of the metal implant in Example 6;

[0044] Figure 2 are the surface morphology diagrams and energy spectrum diagrams of the metal implants in Example 2, Example 3, Example 4, and Example 5 of the present invention; wherein, (a) and (e) are respectively the surface morphology diagram and energy spectrum diagram of Example 2, (b) and (f) are respectively the surface morphology diagram and energy spectrum diagram of Example 3, (c) and (g) are respectively the surface morphology diagram and energy spectrum diagram of Example 4, and (d) and (h) are the surface morphology diagram and energy spectrum diagram of Example 5;

[0045] Figure 3Surface topography and energy spectrum diagrams of the metal implants of Examples 7, 8, 9, and 10 of the present invention; wherein, (a) and (e) are respectively the surface topography and energy spectrum diagrams of Example 7, (b) and (f) are respectively the surface topography and energy spectrum diagrams of Example 8, (c) and (g) are respectively the surface topography and energy spectrum diagrams of Example 9, and (d) and (h) are the surface topography and energy spectrum diagrams of Example 10;

[0046] Figure 4 Cell survival rates of the leaching solutions of the examples of the present invention co-incubated with MC3T3-E1 cells for 72 h; wherein, (a) is the result of Example 2, (b) is the result of Example 3, (c) is the result of Example 4, (d) is the result of Example 5, (e) is the result of Example 7, (f) is the result of Example 8, (g) is the result of Example 9, (h) is the result of Example 10, (i) is the result of Example 11, and (j) is the result of Comparative Example 1;

[0047] Figure 5 Live / dead cell staining results of the leaching solutions of the examples of the present invention co-incubated with MC3T3-E1 cells for 72 h; wherein, (a) is the result of Example 7, (b) is the result of Example 8, (c) is the result of Example 9, and (d) is the result of Example 10;

[0048] Figure 6 Bacteriostatic rate results of the examples of the present invention against Escherichia coli; wherein, (a) is the result of Example 2, (b) is the result of Example 3, (c) is the result of Example 4, (d) is the result of Example 5, (e) is the result of Example 7, (f) is the result of Example 8, (g) is the result of Example 9, (h) is the result of Example 10, (i) is the result of Example 11, and (j) is the result of Comparative Example 1;

[0049] Figure 7 Plate coating counting test results of the examples of the present invention; wherein, (a) is the result of Example 7, (b) is the result of Example 8, (c) is the result of Example 9, and (d) is the result of Example 10. Detailed implementation manners

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific examples.

[0051] Example 1

[0052] The pore size of the porous titanium alloy is set at 640 μm, the rod diameter is set at 0.32 mm, the theoretical porosity is 71.64%, and it has a simple cubic unit structure. The STL format file of the structural model is imported into the computer, and the generated scanning path file is imported into the control software of the printer to prepare the porous titanium alloy using a laser printer. The SLM processing method in the laser printer is used to prepare the porous titanium alloy. Specifically as follows: laser power 160 W, scanning speed 1100 mm / s, scanning spacing 80 μm, layer thickness 30 μm.

[0053] The formed porous titanium alloy with a volume of 11.84 mm * 11.84 mm * 11.84 mm is ultrasonically cleaned successively with absolute ethanol and deionized water, and chemically polished to clean the surface contamination layer and residual powder of the porous titanium alloy, and then air-dried for standby. The chemically polished sample is placed in a laboratory vacuum box furnace and annealed at 800 °C for 2 h, and then cooled with the furnace and stored for standby.

[0054] Example 2

[0055] The ultrasonically cleaned sample of Example 1 is used as the working electrode and connected to the anode of the micro-arc oxidation power supply, and the stainless steel sheet is connected to the cathode of the power supply and placed in the electrolyte. Micro-arc oxidation is carried out in a constant voltage mode, with a current density of 0.1 A / cm 2 , a positive voltage of 400 V, a negative voltage of 0, a positive pulse number of 1, a negative pulse number of 1, a positive duty cycle of 30%, a negative duty cycle of 20%, a frequency of 600 Hz, and a time of 10 min. The electrolyte system uses 0.15 mol / L calcium silicate (CaSiO 3 ) as the calcium source, 0.00833 mol / L sodium hexametaphosphate (NaPO 3 ) 6 as the phosphorus source, 5 g / L sodium hydroxide (NaOH), and 5 ml / L glycerol (C 3 H 8 O 3 ) as an auxiliary additive. The prepared sample is cleaned and dried for standby.

[0056] Example 3

[0057] The ultrasonically cleaned sample of Example 1 is used as the working electrode and connected to the anode of the micro-arc oxidation power supply, and the stainless steel sheet is connected to the cathode of the power supply and placed in the electrolyte. Micro-arc oxidation is carried out in a constant voltage mode, with a current density of 0.1 A / cm 2 , a positive voltage of 400 V, a negative voltage of 0, a positive pulse number of 1, a negative pulse number of 1, a positive duty cycle of 30%, a negative duty cycle of 20%, a frequency of 600 Hz, and a time of 10 min. The electrolyte system uses 0.15 mol / L calcium silicate (CaSiO 3 ) as the calcium source, 0.00833 mol / L sodium hexametaphosphate (NaPO 3 )6 As a phosphorus source, 5 mmol / L strontium chloride (SrCl 2 ·6H 2 O) is used as the source of functional elements, 5 g / L sodium hydroxide (NaOH), and 5 ml / L glycerol (C 3 H 8 O 3 ) is used as an auxiliary additive. The prepared samples are cleaned and dried for later use.

[0058] Example 4

[0059] The sample of Example 1 that has been ultrasonically cleaned is used as the working electrode and connected to the anode of the micro-arc oxidation power supply. The stainless steel sheet is connected to the cathode of the power supply and placed in the electrolyte. Micro-arc oxidation is carried out in a constant voltage mode, with a current density of 0.1 A / cm 2 , a positive voltage of 400 V, a negative voltage of 0, a positive pulse number of 1, a negative pulse number of 1, a positive duty cycle of 30%, a negative duty cycle of 20%, a frequency of 600 Hz, and a time of 10 min. The electrolyte system uses 0.15 mol / L calcium silicate (CaSiO 3 ) as the calcium source, 0.00833 mol / L sodium hexametaphosphate (NaPO 3 ) 6 as the phosphorus source, 5 mmol / L copper acetate (C 4 H 6 CuO 4 ) as the source of functional elements, 5 g / L sodium hydroxide (NaOH), and 5 ml / L glycerol (C 3 H 8 O 3 ) as the auxiliary additive. The prepared samples are cleaned and dried for later use.

[0060] Example 5

[0061] The sample of Example 1 that has been ultrasonically cleaned is used as the working electrode and connected to the anode of the micro-arc oxidation power supply. The stainless steel sheet is connected to the cathode of the power supply and placed in the electrolyte. Micro-arc oxidation is carried out in a constant voltage mode, with a current density of 0.1 A / cm 2 , a positive voltage of 400 V, a negative voltage of 0, a positive pulse number of 1, a negative pulse number of 1, a positive duty cycle of 30%, a negative duty cycle of 20%, a frequency of 600 Hz, and a time of 10 min. The electrolyte system uses 0.15 mol / L calcium silicate (CaSiO 3 ) as the calcium source, 0.00833 mol / L sodium hexametaphosphate (NaPO 3 ) 6 as the phosphorus source, 5 mmol / L copper acetate (C 4 H 6 CuO 4 ), 5 mmol / L strontium chloride (SrCl2 ·6H 2 O) as the source of functional elements, 5 g / L sodium hydroxide (NaOH), 5 ml / L glycerol (C 3 H 8 O 3 ) is the auxiliary additive. The prepared samples are cleaned and dried for later use.

[0062] Example 6

[0063] Put the sample of Example 2 into the inner liner of the hydrothermal reactor for later use. Take 50 mL of deionized water, add ammonia water to adjust the pH of the solution to 10, and add the solution to the inner liner of the hydrothermal reactor containing the micro-arc oxidation coating sample. Then seal the reactor and place it in an oven at 180 °C for 6 h. After the reaction, cool it to room temperature and take out the sample. Clean and dry it ultrasonically with deionized water and absolute ethanol in turn for later use.

[0064] Example 7

[0065] Put the sample of Example 2 into the inner liner of the hydrothermal reactor for later use. Take 50 mL of deionized water, add ammonia water to adjust the pH of the solution to 10 as the basic medium for the hydrothermal reaction. Weigh 0.125 mmol of strontium chloride (SrCl 2 ·6H 2 O) as the additive, ultrasonically dissolve it, and then add the mixed solution to the inner liner of the hydrothermal reactor containing the micro-arc oxidation coating sample. Then seal the reactor and place it in an oven at 180 °C for 6 h. After the reaction, cool it to room temperature and take out the sample. Clean and dry it ultrasonically with deionized water and absolute ethanol in turn for later use.

[0066] Example 8

[0067] Put the sample of Example 2 into the inner liner of the hydrothermal reactor for later use. Take 50 mL of deionized water, add ammonia water to adjust the pH of the solution to 10 as the basic medium for the hydrothermal reaction. Weigh 0.125 mmol of strontium chloride (SrCl 2 ·6H 2 O), 0.0625 mmol of copper acetate (C 4 H 6 CuO 4 ) as the additive, ultrasonically dissolve it, and then add the mixed solution to the inner liner of the hydrothermal reactor containing the micro-arc oxidation coating sample. Then seal the reactor and place it in an oven at 180 °C for 6 h. After the reaction, cool it to room temperature and take out the sample. Clean and dry it ultrasonically with deionized water and absolute ethanol in turn for later use.

[0068] Example 9

[0069] Put the sample of Example 2 into the inner liner of the hydrothermal reactor for standby. Take 50 mL of deionized water, add ammonia water dropwise to adjust the pH of the solution to 10 as the basic medium for hydrothermal reaction. Weigh 0.125 mmol of strontium chloride (SrCl 2 ·6H 2 O), 0.125 mmol of copper acetate (C 4 H 6 CuO 4 ) as additives. After ultrasonic dissolution, add the mixed solution to the inner liner of the hydrothermal reactor containing the micro-arc oxidation coating sample. Then seal the reactor and place it in an oven at 180 °C for reaction for 6 h. After the reaction, cool it to room temperature, take out the sample, and ultrasonically clean and dry it with deionized water and absolute ethanol in sequence for standby.

[0070] Example 10

[0071] Put the sample of Example 2 into the inner liner of the hydrothermal reactor for standby. Take 50 mL of deionized water, add ammonia water dropwise to adjust the pH of the solution to 10 as the basic medium for hydrothermal reaction. Weigh 0.125 mmol of strontium chloride (SrCl 2 ·6H 2 O), 0.25 mmol of copper acetate (C 4 H 6 CuO 4 ) as additives. After ultrasonic dissolution, add the mixed solution to the inner liner of the hydrothermal reactor containing the micro-arc oxidation coating sample. Then seal the reactor and place it in an oven at 180 °C for reaction for 6 h. After the reaction, cool it to room temperature, take out the sample, and ultrasonically clean and dry it with deionized water and absolute ethanol in sequence for standby.

[0072] Example 11

[0073] Put the sample of Example 5 into the inner liner of the hydrothermal reactor for standby. Take 50 mL of deionized water, add ammonia water dropwise to adjust the pH of the solution to 10 as the basic medium for hydrothermal reaction. Weigh 0.125 mmol of strontium chloride (SrCl 2 ·6H 2 O), 0.0625 mmol of copper acetate (C 4 H 6 CuO 4 ) as additives. After ultrasonic dissolution, add the mixed solution to the inner liner of the hydrothermal reactor containing the micro-arc oxidation coating sample. Then seal the reactor and place it in an oven at 180 °C for reaction for 6 h. After the reaction, cool it to room temperature, take out the sample, and ultrasonically clean and dry it with deionized water and absolute ethanol in sequence for standby.

[0074] Comparative Example 1

[0075] Put the sample of Example 8 into a vacuum heat treatment furnace, keep it at 500 °C for 2 h, and then cool it to room temperature in the furnace and take out the sample for standby.

[0076] Figure 1 The surface morphologies of the metal implants of Example 1, Example 2, and Example 6 are shown. Among them, (a) is the surface morphology of the metal implant of Example 1, (b) is the surface morphology of the metal implant of Example 2, and (c) is the surface morphology of the metal implant of Example 6. It can be seen from the figure that the surfaces of the coatings after micro-arc oxidation and micro-arc oxidation-hydrothermal treatment are rougher. The increased roughness can provide more attachment sites for cell adhesion and proliferation, which helps to improve the stability of the implant in the body.

[0077] Figure 2 The surface morphologies and energy spectra of the metal implants of Example 2, Example 3, Example 4, and Example 5 are shown. Among them, (a) and (e) are the surface morphology diagram and energy spectrum diagram of Example 2 respectively, (b) and (f) are the surface morphology diagram and energy spectrum diagram of Example 3 respectively, (c) and (g) are the surface morphology diagram and energy spectrum diagram of Example 4 respectively, and (d) and (h) are the surface morphology diagram and energy spectrum diagram of Example 5 respectively. It can be seen from the figure that the honeycomb morphology of micro-arc oxidation can be clearly observed in Example 2 and Example 4, and the surface roughness increases and the number of micropores decreases on the surfaces of Example 3 and Example 5.

[0078] Table 1 Surface composition analysis of the metal implants of Example 2, Example 3, Example 4, and Example 5

[0079]

[0080] It can be seen from Table 1 that the corresponding doping elements are detected in the micro-arc oxidation coatings.

[0081] Figure 3 The surface morphologies and energy spectra of the metal implants of Example 7, Example 8, Example 9, and Example 10 are shown. Among them, (a) and (e) are the surface morphology diagram and energy spectrum diagram of Example 7 respectively, (b) and (f) are the surface morphology diagram and energy spectrum diagram of Example 8 respectively, (c) and (g) are the surface morphology diagram and energy spectrum diagram of Example 9 respectively, and (d) and (h) are the surface morphology diagram and energy spectrum diagram of Example 10 respectively. It can be seen from the figure that the honeycomb morphology of micro-arc oxidation can be clearly observed in Example 7 and Example 8. With the increase of the copper content in the hydrothermal reaction system, the surface of the example becomes rough and uneven, the honeycomb morphology disappears, and the micropores decrease.

[0082] Table 2 Surface composition analysis of the metal implants of Example 7, Example 8, Example 9, and Example 10:

[0083]

[0084] As can be seen from Table 2, with the increase of Cu content in the hydrothermal reaction system, the Cu content in the coating also gradually increases.

[0085] Performance test:

[0086] (1) Detect cell viability by CCK-8 assay

[0087] Autoclave the samples of the examples at 121 °C for 20 min, dry them overnight at 60 °C after autoclaving, and leach them in DMEM complete medium at an extraction ratio of 1.25 cm 2 / mL, 95% relative humidity, 5% CO 2 and at 37 °C for 72 h to obtain the leachate of the corresponding samples.

[0088] Digest MC3T3-E1 cells with 0.25% trypsin (containing EDTA) to prepare a single-cell suspension, centrifuge it at 1000 rpm for 5 min, resuspend the cells with DMEM medium containing 10% fetal bovine serum, and adjust the cell density to 3×10 4 cells / mL. Inoculate the above cell suspension into a 96-well plate, inoculate 0.2 mL per well, set up a negative control and a test group, with 3 wells in each group, and place it at 37 °C, 5% CO 2 incubator and culture for 24 h.

[0089] Take the 96-well culture plate inoculated and cultured for 24 h, aspirate the liquid in the wells, add the leaching medium to the negative control group, and add the original leaching solution of the example to the test group, 0.2 mL per well, and place it in 5% CO 2 incubator and continue to culture at 37 °C ± 2 °C for 72 h. Take the 96-well plate after co-culturing for 72 h, aspirate the liquid in the wells, wash each well once with phosphate buffer solution, add the medium containing 10% CCK-8 at 0.1 mL / well, and continue to culture at 5% CO 2 and 37 °C for 2 h. Then detect and record the absorbance value of each well at a wavelength of 450 nm by an enzyme-linked immunosorbent assay. Calculate the relative activity of each group of cells according to formula (1).

[0090]

[0091] Among them, the background OD value is the absorbance of adding only CCK-8 reagent and medium; the blank group OD value is the absorbance of the blank control group; the test group OD value is the absorbance of the test group.

[0092] Figure 4This is the cell survival rate after co-incubating the extraction solution of the embodiments of the present invention with MC3T3-E1 cells for 72 h. Among them, (a) is the result of Example 2, (b) is the result of Example 3, (c) is the result of Example 4, (d) is the result of Example 5, (e) is the result of Example 7, (f) is the result of Example 8, (g) is the result of Example 9, (h) is the result of Example 10, (i) is the result of Example 11, and (j) is the result of Comparative Example 1. The results are as Figure 4 shown. After micro-arc oxidation treatment of porous titanium alloy, the cell survival rate of the Sr-doped coating is relatively high, while that of the Cu and Sr co-doped coating is relatively low. When the micro-arc oxidation coating is further hydrothermally treated, the cell survival rate increases significantly. When introducing Cu and Sr elements, with the increase of the copper ion content in the hydrothermal reaction system in the embodiments, the cell survival rate gradually decreases, which is related to the coating composition and microstructure. Example 11 after hydrothermal treatment of the micro-arc oxidation coating doped with Cu and Sr elements as the substrate in the hydrothermal system containing Cu and Sr elements has a significantly higher cell survival rate than that of Example 5 and is not much different from that of Example 8. Comparative Example 1 is the sample after heat treatment of Example 8, and its cell survival rate is equivalent to that of Example 8.

[0093] (2) Live / dead cell staining experiment

[0094] Autoclave the samples of the embodiments at 121 °C for 20 min, dry them overnight at 60 °C after autoclaving, and extract for 72 h in DMEM complete medium, 95% relative humidity, 5% CO 2 at an extraction ratio of 1.25 cm 2 / mL, 5% CO

[0095] and 37 °C environment to obtain the extraction solution of the corresponding samples. 4 Digest MC3T3-E1 cells with 0.25% trypsin (containing EDTA) to prepare a single cell suspension, centrifuge at 1000 rpm for 5 min, resuspend the cells with DMEM medium containing 10% fetal bovine serum, and adjust the cell density to 4×10 2 cells / mL. Inoculate the above cell suspension into a 24-well plate, 1 mL per well, set up a negative control and experimental groups, and culture at 37 °C, 5% CO 2 in an incubator for 24 h. Take the 24-well culture plate inoculated and cultured for 24 h, aspirate the liquid in the wells, add the extraction medium to the negative control group, and add the original extraction solution of the embodiments to the experimental groups, 1 mL per well, and continue to culture at 37 °C ± 2 °C in a 5% CO 2 incubator for 72 h. Take the 24-well culture plate inoculated and cultured for 24 h, aspirate the liquid in the wells, add the extraction medium to the negative control group, and add the original extraction solution of the embodiments to the experimental groups, 1 mL per well, and continue to culture at 37 °C ± 2 °C in a 5% CO

[0096] Dilute reagent A (Calcein-AM) and reagent B (PI) 10 times respectively with the dye diluent (solution C); take 985.5 μL of serum-free medium, mix it with 10 μL of the diluted reagent A and 4.5 μL of reagent B to obtain the staining working solution, and use it immediately after preparation. Take the 24-well plate after co-culture for 72 h, aspirate the liquid in the wells, and wash each well once with phosphate buffer. Add 1 mL of the staining working solution to each well, incubate in the dark at 4 °C for 15 min, and then observe with a fluorescence microscope.

[0097] Figure 5 This is the live / dead cell staining result of the leaching solution of the embodiment of the present invention co-incubated with MC3T3-E1 cells for 72 h; among them, (a) is the result of Example 7, (b) is the result of Example 8, (c) is the result of Example 9, and (d) is the result of Example 10. The results are as Figure 5 shown. In the coatings treated by micro-arc oxidation and micro-arc oxidation-hydrothermal treatment, not only are the MC3T3-E1 cells more complete in morphology, but also the survival rate is relatively high.

[0098] (3) Antibacterial experiment

[0099] LB liquid medium: Measure 100 mL of distilled water with a graduated cylinder and pour it into a 250 mL reagent bottle. Weigh 2.5 g of LB broth medium with an analytical electronic balance and add it to the mixture. After mixing, sterilize it at 121 °C for 15 min in a high-temperature high-pressure steam sterilizer and set it aside for use.

[0100] LB solid medium: Measure 100 mL of distilled water with a graduated cylinder and pour it into a 250 mL reagent bottle. Weigh 2.5 g of LB broth medium and 1.5 g of agar powder with an analytical electronic balance. Add the weighed reagents to the mixture and mix well. Then sterilize it at 121 °C for 15 min in a high-temperature high-pressure steam sterilizer. Wait for the medium to cool to about 40 - 50 °C, and use an electric pipette to aspirate 15 mL of the medium and pour it into a disposable sterile petri dish.

[0101] Take a 12 mL bacterial culture tube, add 3 mL of LB liquid medium, pick a single colony from the solid medium of Escherichia coli and add it to the liquid medium, and place it in a constant temperature oscillator (37 °C, 200 rpm) and shake it overnight (15 h).

[0102] Put the samples of the examples into 12 mL bacterial culture tubes according to the grouping, and sterilize them at 121 °C for 20 min in a high-temperature high-pressure steam sterilizer for standby.

[0103] Add LB liquid medium to the sample tubes of the examples at a ratio of 0.2 g / mL and leach for 72 h. Dilute the bacterial solution to 10 with LB medium 8CFU / mL. Take 1 mL of the diluted bacterial solution, centrifuge at 8000 rpm for 3 min, and discard the supernatant. Take 1 mL of the extract solution of the sample in the example to resuspend the bacterial precipitate and dilute it 100 times. Place it in a constant temperature incubator at 37 °C and let it stand for 24 h.

[0104] After the cultivation is completed, serially dilute the bacterial solution 10-fold with sterile PBS solution. Take 100 μL of the diluted solution and evenly coat it on the LB solid medium. Place it in a constant temperature incubator at 37 °C for 18 h, take it out, take a photo and record the number of colonies.

[0105] Calculation method of the bacterial solution concentration (CFU / mL): number of colonies × dilution factor × 10 (coated with 0.1 mL)

[0106] Antibacterial rate = (1 - concentration of the experimental group bacterial solution / concentration of the control group bacterial solution) × 100%

[0107] Taking the micro-arc oxidation calcium phosphate film layer as the control group, the results are as Figure 6 and 7 described, Figure 6 are the antibacterial rate results of the examples of the present invention against Escherichia coli; among them, (a) is the result of Example 2, (b) is the result of Example 3, (c) is the result of Example 4, (d) is the result of Example 5, (e) is the result of Example 7, (f) is the result of Example 8, (g) is the result of Example 9, (h) is the result of Example 10, (i) is the result of Example 11, and (j) is the result of Comparative Example 1; Figure 7 are the plate coating counting test results of the examples of the present invention; among them, (a) is the result of Example 7, (b) is the result of Example 8, (c) is the result of Example 9, and (d) is the result of Example 10.

[0108] The results show that after culturing for 24 hours at 37 °C, the antibacterial rate of the examples of micro-arc oxidation hydrothermal treatment is significantly improved. The Cu, Sr co-doped coating has a high antibacterial rate. As the copper content increases, the antibacterial rate decreases, which is related to the composition and microstructure of the coating. Example 11 after hydrothermal treatment in a hydrothermal system containing Cu and Sr elements with the micro-arc oxidation coating doped with Cu and Sr elements as the substrate has a slightly higher antibacterial rate than Example 5, but is significantly lower than that of Example 8. Comparative Example 1 is the heat treatment of Example 8. Although the cell survival rate is equivalent to that of Example 8, the antibacterial property has decreased.

[0109] In summary, the optimal example is Example 8, which realizes high cell survival rate and high antibacterial property.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A titanium-based implant having both antibacterial and osteogenic properties, characterized in that: It includes a porous titanium alloy substrate and a coating formed on the porous titanium alloy substrate; Wherein, the coating is a composite coating doped with functional elements obtained by micro-arc oxidation and hydrothermal treatment; The functional element is one or more of strontium, copper, zinc, magnesium and silver.

2. The titanium-based implant according to claim 1, characterized in that The functional elements are strontium and copper; Preferably, the porous titanium alloy has a pore size of 200 to 700 μm, a rod diameter of 200 to 400 μm, and a porosity of 60 to 90%; Preferably, the functional element doping is doping the functional element during micro-arc oxidation and / or doping the functional element during hydrothermal treatment; further preferably, the functional element is doped during hydrothermal treatment.

3. A method for preparing a titanium-based implant having both antibacterial and osteogenic properties as claimed in claim 1 or 2, characterized in that: The following steps are involved: The pretreated porous titanium alloy is used as a substrate, and a porous titanium alloy loaded with a micro-arc oxidation coating is obtained by micro-arc oxidation treatment. Using porous titanium alloy loaded with micro-arc oxidation coating as the matrix, hydrothermal treatment was used to prepare the composite coating, thus completing the preparation of titanium-based implants with both antibacterial and osteogenic properties.

4. The preparation method according to claim 3, characterized in that: The pretreatment includes cleaning, polishing and heat treatment of the porous titanium alloy; Preferably, the cleaning is ultrasonic cleaning; Preferably, the polishing is a chemical polishing process; Preferably, the heat treatment is an annealing treatment at 700-900° C. for 1-4 hours.

5. The preparation method according to claim 3, characterized in that: The micro-arc oxidation treatment comprises: connecting the pretreated porous titanium alloy as a working electrode to the anode of a micro-arc oxidation power supply, connecting a stainless steel sheet to the cathode of the power supply, and placing the stainless steel sheet in an electrolyte; Preferably, the micro-arc oxidation adopts a constant voltage mode with a current density of 0.05-0.15A / cm 2 , forward voltage 400-450V, negative voltage 0, number of positive pulses 1, number of negative pulses 1, positive duty cycle 25-35%, negative duty cycle 15-25%, frequency 500-700Hz, time 10~15min.

6. The preparation method according to claim 5, characterized in that: The electrolyte includes a calcium source, a phosphorus source, and auxiliary additives; Preferably, the calcium source is calcium silicate, and the concentration of calcium silicate is 0.10-0.20 mol / L; Preferably, the phosphorus source is sodium hexametaphosphate, and the concentration of sodium hexametaphosphate is 0.005-0.015 mol / L; Preferably, the auxiliary additives include sodium hydroxide and glycerol; the concentration of sodium hydroxide is 4-6 g / L; the concentration of glycerol is 4-6 ml / L.

7. The preparation method according to claim 6, characterized in that: The electrolyte also includes an additive containing a functional element; Preferably, the additive containing functional elements includes strontium chloride and / or copper acetate; Preferably, the concentrations of strontium chloride and copper acetate are both 1-6 mmol / L.

8. The preparation method according to claim 3, characterized in that: The hydrothermal treatment comprises placing the porous titanium alloy loaded with the micro-arc oxidation coating in a reaction medium and reacting at 170-190° C. for 4-8 hours; Preferably, the reaction medium comprises a reaction base medium, the reaction base medium comprises water, and the pH of the aqueous solution is adjusted to 9-11 using alkali.

9. The preparation method according to claim 8, characterized in that: The reaction medium further comprises an additive containing a functional element; the additive containing the functional element comprises strontium chloride and / or copper acetate; Preferably, the ratio of water, strontium chloride and copper acetate is (40-60 mL):(0.05-0.30 mmol):(0.05-0.30 mmol), preferably 50 mL:(0.05-0.2 mmol):(0.05-0.15 mmol).

10. Use of the titanium-based implant with both antibacterial and osteogenic properties according to claim 1 or 2 or the titanium-based implant with both antibacterial and osteogenic properties obtained by the preparation method according to any one of claims 3 to 9 in the field of bone tissue repair and bone defect filling.

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